Topical nanoparticulate spironolactone formulation

ABSTRACT

The invention relates to a topical nanoparticulate spironolactone formulation comprising nanoparticles having a mean diameter, measured by a photon correlation spectroscopy, in the range of from about 300 nm to about 900 nm. The nanoparticles are incorporated into a crystalline network system comprising a dispersion of solid crystals of polar lipids, said lipids exposing their hydrophilic side outwards and their hydrophobic side inwards towards the spironolactone nanoparticles.

RELATED APPLICATIONS

This application is a is a national stage application, filed under 35U.S.C. §371, of International Application No. PCT/GB02/05680, filed onDec. 13, 2002.

The present invention relates to the use of spironolactone in the formof nanoparticles in the topical treatment of a condition responding toanti-androgens. Such conditions include acne, hirsutism, androgenicalopecia or rosacea.

Spironolactone is known as an aldosterone inhibitor having utility as apotassium sparing diuretic. It is commercially available as e.g.aldactone and may be employed e.g. in the treatment of congestive heartfailure. Spironolactone has extremely low solubility in water, viz: 2.8mg/100 ml. This low solubility can adversely affect absorption of thedrug substance in vivo, leading to poor bioavailability. Consequentlyhigher amounts of the drug substance are required to achieve the desiredblood levels. The poor solubility of spironolactone also restricts theoptions available for formulating the drug substance.

Other pharmaceutical applications make use of the anti-androgeniceffects of Spironolactone for the treatment of a variety of skindisorders such as acne, hirsutism, androgenic alopecia and rosacea.Topical administration for these disorders would be the preferred routedue to the greatly reduced systemic side effects. However, again it isthe poor solubility of the drug, which limits the development ofefficacious and aesthetically acceptable topical formulations.

Following oral administration, the absorption of drugs from theintestine is mainly dependent on their solubility in the intestinalfluids and their intestinal permeability. Poorly soluble drugs generallyhave low dissolution rates and exhibit only a small concentrationgradient across the intestinal mucosa, which can result in low andunreliable levels of absorption. Drug substances which have lowsolubility also suffer from disadvantages in respect of other routes ofadministration, for example, topically.

Significant efforts have been directed to producing drug substances inthe form of microparticles and nanoparticles. However, preparation ofsuch small particles is not a trivial matter and can give rise tofurther difficulties both in relation to technical aspects of theprocess and in obtaining a satisfactory product. Thus for example therecan be difficulties, especially on a manufacturing scale in obtaining aconsistent and narrow particle size range. Furthermore, it is necessaryto obtain stable products, e.g. nanosuspensions, but microparticles andnanoparticles have a tendency to aggregate and flocculate, which hasadverse consequences for the stability of the product A number ofdifferent approaches have been investigated for the preparation ofmicroparticles and nanoparticles.

U.S. Pat. No. 5,091,188 describes a method for preparing injectablesolutions of water-insoluble drugs, which comprises reducing thecrystalline drug substance to dimensions in the range 50 nm to 10 μm, bysonication or other processes inducing high shear, in the presence of aphospholipid or other membrane-forming amphipathic lipid, whereby thedrug microcrystals become coated with said lipid.

U.S. Pat. No 5,145,684 describes particles of crystalline drug substancehaving a non-cross linked surface modifier adsorbed on the surface andan effective average particle size of less than about 400 nm. Theseparticles are said to be prepared by milling in the presence of grindingmedia, using for example a ball mill, an attrition mill, a vibratorymill or a media mill.

International Patent Application WO 96/14830 (U.S. Pat. No. 5,858,410)describes a drug carrier which comprises particles of a pure activecompound which is insoluble or only sparingly soluble in water, whichhas an average diameter of 10 nm to 1,000 nm and the proportion ofparticles larger than 5 μm in the total population is less than 0.1%.Preparation of the particles, with or preferably without surfactant, bymeans of cavitation (e.g. using a piston-gap homogenizer) or by shearingand impact forces (i.e. the jet stream principle) is also described.

There is a need for a topical formulation of nanoparticulatespironolactone that overcomes the problems of formulating the drug fortopical administration.

The applicants have now shown that for topical administration, thespironolactone in the form of nanoparticles can be successfullyincorporated into a cream base consisting of a crystalline network ofmonoglycerides in water.

In a first aspect therefore the present invention provides a topicalnanoparticulate spironolactone formulation comprising nanoparticleshaving a mean diameter, measured by photon correlation spectroscopy, inthe range of from about 300 nm to about 900 nm, preferably 400 nm to 600nm incorporated into a crystalline network system comprising adispersion of solid crystals of polar lipids, said lipids exposing theirhydrophilic side outwards and their hydrophobic side inwards towards thespironolactone nanoparticles.

The formulation is suitable for application to the skin for use intreating dermatological conditions known to be treatable withantiandrogens e.g. acne, androgenic alopecia, hirsutism and rosacea.Cream bases consisting of a crystalline network of monoglycerides aredescribed in WO87/02582, WO82/03173 and WO93/20812. Examples of suchcrystalline networks of monoglycerides are known as Crystalip™.

The lipids may have a crystallisation temperature of between 20° C. and100° C. Preferable lipid crystals are β-crystals from a monoglyceride ofa fatty acid having a chain length of 12-18 carbon atoms or monoglycerolethers having ether chains of the corresponding length or fatty acidesters of ascorbic acid with a fatty acid chain length of 12-18 carbonatoms or mixtures thereof. The fatty acids as well as the ethers may besaturated or unsaturated, preferably saturated ones.

The fatty acids may therefore include lauric acid (C₁₂), myristic acid(C₁₄), palmitic acid (C₁₆) or stearic acid (C₁₈), although C₁₃, C₁₅, orC₁₇ acids could also be used.

Preferable monoglycerides may be a 1- or 2-monoglyceride, preferably a1-monolaurin, 1-monomyristin, 1-monopalmitin and 1-monostearin or amixture of two or more of these such as a mixture of 1-monolaurin and1-monomyristin. Examples of unsaturated monoglycerides aremonopalmitolein, monoolein, monolinolein and monoliniolenin.

The composition consists essentially of a dispersion of the above lipidcrystals in water or any other polar liquid or mixtures thereof havingthe ability to allow crystal formation. Examples of polar lipids for usein accordance with the invention are water, glycerol, propylene glycoland ethylene glycol or mixtures thereof, however other suitable polarlipids may also be used.

The spironolactone is protected within the network up to the time of usebut upon application to the skin, the spironolactone comes into contactwith the skin surface as a consequence of softening or melting of thecrystalline structure of the shell.

Generally one would expect a noticeable increase in particle size onstorage following the incorporation of very fine solid particles into amatrix which contains hydrophilic as well as lipophilic structures.Surprisingly, this did not happen and there was no noticeable crystalgrowth of Spironolactone over a seven month period. Furthermore, thecream has shown an increased flux rate in a membrane model with respectto a cream with non-nanoparticulate spironolactone.

As is well known in the pharmaceutical art, particle size may bemeasured by a variety of methods, which can give rise to apparentlydifferent reported particle sizes. Such methods include photoncorrelation spectroscopy (PCS) and laser diffraction. Furthermore theparticle size may be reported as an average particle size (e.g. a numberaverage, weight average or volume average particle size). In the presentspecification, unless indicated otherwise, the particle size will bequoted as a volume average particle size. Thus for example, a D₅₀ of 500nm indicates that 50% by volume of the particles have a diameter of lessthan 500 nm. Alternatively it can be stated that the particles having adiameter of less than 500 nm occupy 50% of the total volume occupied bythe total number of particles.

When the particle size of spironolactone according to the presentinvention is measured by laser diffraction the D₅₀ is in the range350-750 nm and the D₉₉ is in the range 500-900 nm.

Nanosuspensions and nanoparticles comprising spironolactone according tothe present invention preferably incorporate a stabiliser to preventaggregation of the nanoparticles. Such stabilisers, which are well knownin the art, are described in more detail hereinafter.

In this specification nanoparticles comprising spironolactone andnanosuspensions comprising spironolactone according to the presentinvention will be referred to as nanoparticulate spironolactone. Itshould be appreciated that this term also includes nanoparticles andnanosuspensions comprising spironolactone in association with astabiliser.

Nanoparticulate spironolactone according to the invention, may beprepared by any known method for the preparation of nanoparticles, inparticular by high pressure homogenisation.

The nanoparticulate spironolactone may be prepared by subjecting acoarse dispersion of spironolactone to cavitation. Preferably thenanoparticles are prepared using a high pressure piston-gap homogeniser.The nanoparticles may be associated with a stabiliser. Such stabilisers,which are well known in the art, are described in more detailhereinafter.

For the preparation of nanoparticles it is preferred that thespironolactone starting material be utilised in the form of coarseparticles, preferably having a particle size of less than about 100 μm.If necessary, the particle size of the spironolactone may be reduced tothis level by conventional means, such as milling. The coarse particlesof spironolactone are preferably dispersed in a liquid medium comprisinga solvent in which the drug substance is essentially insoluble. In thecase of spironolactone the liquid medium preferably comprises an aqueoussolvent and most preferably consists essentially of water. Theconcentration of spironolactone in the said dispersion of coarseparticles may be in the range 0.1 to 50%. The coarse dispersion may thenbe utilised in any known method for obtaining nanoparticles.

A preferred method is high pressure homogenization, wherein particlesize is reduced mainly by cavitation. This is most preferably achievedusing a high-pressure piston-gap homogeniser. In this method, thedispersion of coarse particles is forced at a high flow rate through agap which is approximately 25 μm wide. The static pressure exerted onthe liquid falls below the vapour pressure of the liquid. The liquidtherefore boils, resulting in the formation of gas bubbles within thearea of the gap. However, once the liquid exits from the gap, normalpressure prevails and the gas bubbles collapse. The powerful implosionforces which result are strong enough to break up the coarse particlesof drug substance, resulting in the formation of nanoparticles.

High pressure homogenisation may be carried out at a pressure in therange 100 to 3000 bar, preferably 1000 to 2000 bar (10⁷ to 3×10⁸ Pa,preferably 10⁸ to 2×10⁸ Pa) and at a temperature in the range 0 to 50°C., preferably 10 to 20° C., e.g. around 15° C. The homogenisation maybe carried out in a series of cycles until the desired particle size isobtained, or as a continuous process, e.g. over a period of 2-30 hours,preferably 2-10 hours.

Nanosuspensions of spironolactone according to the present inventionpreferably incorporate a stabiliser to prevent aggregation of thenanoparticles. Said stabiliser may be introduced at any suitable stageduring the manufacture of the nanosuspension. Thus for example,surfactant may be added to the initial coarse dispersion prior to theformation of nanoparticles or after reduction of the particles size,e.g. by high pressure homogenization, has taken place. Alternatively aportion of the stabiliser may be added before and a portion after thestep of particle size reduction. Preferably stabiliser is present in thecoarse dispersion. The concentration of stabiliser, either in the coarsedispersion or the nanosuspension may be in the range 0 to 10%.

Stabilisers which may be employed in the preparation of nanosuspensionsaccording to the present invention may be selected from conventionalstabilisers, and may include compounds which are also described asurfactants and surface modifiers. Thus examples of stabiliser which maybe employed include: polyoxyethylene sorbitan fatty acid esters, e.g.Tweens and Spans; polyoxyethylene stearates; polyoxyethylene alkylesters; polyethylene glycols; block polymers and block copolymers suchas poloxamers e.g Lutrol F68, and poloxamines; lecithins of variousorigin (e.g. egg-lecithin or soya-lecithin), chemically-modifiedlecithins (e.g. hydrated lecithin), as well as phospholipids andsphingolipids, sterols (e.g. cholesterin derivatives, as well asstigmasterin), esters and ethers of sugars or sugar alcohols with fattyacids or fatty alcohols (e.g. saccharose monostearate); ethoxylatedmono- and diglycerides, ethoxylated lipids and lipoids, dicetylphosphate, phosphatidyl glycerine, sodium cholate, sodium glycolcholate,sodium taurocholate; sodium citrate; cellulose ethers and celluloseesters (e.g. methyl cellulose, hydroxyethyl cellulose, hydroxypropylcellulose, sodium carboxymethyl cellulose), polyvinyl derivatives suchas polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl acetate,alginates, polyacrylates (e.g. carbopol), xanthanes; pectins, gelatin,casein, gum acacia, cholesterol, tragacanth, stearic acid, calciumstearate, glyceryl monostearate, dioctyl sodium sulfosuccinate (sodiumdocusate); sodium lauryl sulfate, sodium dodecyl sulphate, benzalkoniumchloride, alkyl aryl polyether sulfonate, polyethylene glycols;colloidal silicon dioxide, magnesium aluminium silicate; and phosphates.

A preferred stabiliser is sodium docusate, which is commerciallyavailable as a 70% solution in propylene glycol, under the name Octowet.70PG™ (sodium dioctyl sulfosuccinate).

It will be appreciated from the foregoing that the process is carriedout in a liquid medium and hence the nanoparticulate spironolactoneproduct is initially obtained in the form of a nanosuspension. Ifdesired the liquid medium may be removed, e.g. by lyophilisation orspray drying to provide nanoparticulate spironolactone in solid form. Itwill be appreciated that where a stabiliser is present during themanufacture of a nanosuspension, the corresponding dried nanoparticulateproduct will be associated with said stabiliser.

Following preparation of the nanoparticulate spironolactone, theformulation according to the invention may be prepared as follows. Thepolar lipid is mixed with water and/or any other polar liquid (such asglycerol, ethylene glycol or propylene glycol) having the ability toform crystalline network structures from polar lipids. The mixtureformed has a concentration of water and/or polar liquid, respectively,of 50-95 percent by weight. The mixture is heated to a temperature abovethe transition temperature of the lipid. The transition temperature isdefined as the lowest temperature at which a particle of the lipid incontact with an excess of water or polar liquid absorbs water or polarliquid respectively and is converted into cylindrical or sphericalcrystalline structures having a strong birefringence. The mixture ismaintained above the transition temperature with stirring until theconversion has taken place. The mixture is then cooled with continuedstirring to ambient temperature or the desired temperature, so thatsolid crystalline networks are formed. It is during this cooling down,at a temperature of about 30 to 35° C. that the characteristiccrystalline structure is formed.

The nanoparticulate spironolactone is dispersed in the mixture of polarlipid and water or polar liquid before or while the lipid is transformedinto crystalline structures. To ensure that the nanoparticulatespironolactone is incorporated into the crystalline structure it must beadded before the mixture is cooled below 30 to 35° C.

If the nanoparticulate spironolactone is added after the mixture hasbeen cooled to below 30 to 35° C., a physical mixture is formed but itdoes not form part of the crystalline structure. The nanoparticulatespironolactone therefore does not benefit from protection from andprevention of re-crystallisation and particle size growth of the activecomponent since crystal layers are not formed around the activeparticles.

Nanosuspensions as used in the present invention in the formation of thetopical formulation do not however respond well to heating. Duringheating, agglomerates may be formed and the active component may go intosolution at higher temperatures. This can lead to re-crystallisationduring the cooling down period which can result in a considerableincrease in particle size.

The applicants have however determined a formulation, process andtemperature of incorporation in order to allow formation of thecrystalline structure after the addition of the nanosuspension, whilekeeping the heat exposure of the nanosuspension to a minimum.

Topical nanoparticulate spironolactone formulations according to thepresent invention advantageously incorporate the active drug in the formof a nanosuspension, most preferably in aqueous solution. Pharmaceuticalformulations according to the present invention may be preparedaccording to methods well known in the art.

Topical formulations according to the present invention may be providedas an ointment, cream, gel, liquid, spray or mousse. Aqueouspreparations may contain the nanosuspension as such; non-aqueouspreparations can alternatively comprise dried nanoparticles.

In a second aspect the present invention provides a topicalnanoparticulate spironolactone formulation for use in the topicaltreatment of conditions known to be treatable with antiandrogens, e.g.acne, androgenic alopecia, hirsutism and rosacea.

In a third aspect, the invention provides the use of spironolactonenanosuspensions comprising nanoparticles having a mean diameter,measured by photon correlation spectroscopy, in the range of from about300 nm to about 900 nm, preferably 400 nm to 600 nm in the manufactureof a medicament for the treatment of a condition responding toantiandrogens, such as acne, hirsutism, androgenic alopecia or rosacea.The medicament may be adapted for topical application. The nanoparticlesmay be incorporated into a cream base which may consist of a crystallinenetwork of monoglycerides in water or other polar liquids.

This aspect of the invention extends to providing a method of treating acondition responding to anti androgens comprising administeringnanoparticulate spironolactone formulation as defined above to a patientin need of such treatment. The condition may be acne, hirsutism,androgenic alopecia or rosacea.

In a fourth aspect, the invention provides preparations comprisingcrystalline network system of solid crystals of polar lipids, saidlipids exposing their hydrophilic side outwards and their hydrophobicside inwards towards an incorporated substance for use in the topicaltreatment of acne. The crystalline network system of solid crystals ofpolar lipids have previously been referred to as microcapsules in WO87/02582.

In a fifth aspect there is provided a process for the preparation of atopical nanoparticulate spironolactone formulation comprisingnanoparticles having a mean diameter, measured by photon correlationspectroscopy, in the range of from 300 nm to about 900 nm, wherein theprocess comprises incorporation of a nanosuspension of spironolactoneinto an aqueous dispersion of solid crystals of polar lipids, saidlipids exposing their hydrophilic side outwards and their hydrophobicside inwards towards the spironolactone nanoparticles.

The nanosuspension may be incorporated when the mixture has been cooledto between 60° C. and 35° C., more preferably 55° C. to 45° C., suitably50° C. before the mixture reaches its crystallisation point. Thetemperature of the nanosuspension at incorporation is preferably equalto room temperature i.e. 20 to 25° C.

Preferred features for the second and subsequent aspects of theinvention are as for the first aspect mutatis mutandis.

The invention will now be illustrated with reference to one or more ofthe following non-limiting examples and figures.

FIG. 1 relates to a microscope picture of nanoparticulate spironolactoneaccording to the present invention immediately after it has beenprepared. The scale relates to a distance between each bar of 0.01 mm.

FIG. 2 relates to a microscope picture of nanoparticulate spironolactoneaccording to the present invention after 7 months storage at roomtemperature. The scale relates to a distance between each bar of 0.01mm.

FIG. 3 relates to a microscope picture of commercially availablespironolactone in non-nanoparticulate form. The scale relates to adistance between each bar of 0.01 mm.

FIG. 4 relates to a typical calibration curve of Spironolactonestandards of 0.93-59.2 μg/ml.

FIG. 5 relates to a graph showing the mean flux of spironolactone fromthe nanosuspensions (2% w/w) and aqueous cream (2% w/w) (n=4, mean±SE)y=35.552x−20.258 r2=0.991 Spironolactone aqueous cream 2%y=42.097x−26.784 r2=0.989 Spironolactone nanosuspension 2%

FIG. 6 relates to S. epidermidis mean zone diameter of Crystalip™spironolactone formulation compared to 2% w/w spironolactone in Aqueouscream B.P. (mean±S.D.; n=5)

FIG. 7 relates to P. acnes mean zone diameter of Crystalip™spironolactone formulation compared to 2% spironolactone w/w in Aqueouscream B.P. as comparator (mean±S.D; n=5).

FIG. 8 relates to the particle size of spironolactone nanosuspensionfollowing heating to 50° C. (▪) or 70° C.(□) then cooling compared tounheated nanosuspension (

)

FIG. 9 relates to the particle size of spironolactone nanosuspension 24hours after heating to 50° C. (▪) or 70° C. (□) then cooling compared tounheated nanosuspension (

)

FIG. 10 relates to the viscosity of the mixture following introductionof Octowet™ at 50° C., 70° C. or room temperature.

FIG. 11 relates to the effect of the composition of the mixture on itsviscosity.

EXAMPLES Example 1 Preparation of Nanoparticulate Spironolactone as aTopical Formulation

Preparation of Nanoparticulate Spironolactone.

Table 1 illustrates representative preparations of nanoparticulatespironolactone for incorporation into a crystalline structure inaccordance with the present invention. The nanoparticulatespironolactone may be prepared as follows:

A preparation of an aqueous solution of the stabiliser was incorporatedinto water or buffer for injection under magnetic stirring until a clearsolution was obtained. A slurry was formed by wetting the spironolactonewith the appropriate quantity of the aqueous solution of the surfactant.The resulting suspension was dispersed using a high shear-dispersinginstrument. The suspensions were left under magnetic agitation toeliminate foaming. The resulting suspensions were passed through ahigh-pressure piston gap homogenizer to obtain a nanosuspension.Formulations 1-7 were prepared using an Avestin C5™ and Formulations 8and 9 were prepared using an Avestin C50™. During homogenization thedrug particles are disrupted due to cavitation effects and shear forcesto form small micro-and nanoparticles. The particle sizes weredetermined by photon correlation spectroscopy (PCS) using a Zetasizer3000 HS™ (Malvern). D₅₀ and D₉₀ were measured by laser diffraction usinga Coulter LS230.

TABLE 1 Formulation 1 2 3 4 5 6 7 8 9 Spironolactone % 10 10 20 10 10 1010 10 10 Sodium lauryl 1 — — 0.1 0.4 0.1 — — — sulphate % Lutrol F68 % —1 1 0.4 0.1 0.4 — — — Na Cl — — — — — 0.9 — — — Octowet 70 — — — — — —0.5 0.5 0.5 (sodium (0.35) (0.35) (0.35) docusate) % Water QS to 100%Sample volume 40 40 40 40 40 40 40 100 500 (ml) Results D₅₀ (micron)1.69 0.85 1.06 0.84 0.88 0.86 0.78 0.54 0.539 D₉₀ (micron) 4.39 1.832.49 1.92 1.82 1.5 1.8 0.68 0.772 PCS mean — 581 880 608 681 656 609 415436 diameter PI — 0.7 0.2 0.15 0.03 0.1 0.2 0.05 0.1Preparation of a Crystalip™ Composition

Table 2 illustrates representative preparations of nanoparticulatespironolactone as a topical cream, using formulations 7, 8 or 9 as shownin Table 1. The topical nanosuspension preparations were prepared asfollows:

Water was heated to 70° C. and propylene glycol added. Themonoglycerides were melted at 70° C., and the molten monoglycerides werethen added to the water phase under stirring at 70 rpm. Cooling down ofthe mixture was then started. The stirring speed was increased to 95 rpmwhen the mixture reached around 50° C. when the viscosity increased andthe cold nanosuspension added. The stirring speed was decreased to 75rpm at 35° C. when the β-crystalline structure started to form.

TABLE 2 Formulation A B C D Spironolactone Nanosuspension 20 10 20 20(formulations 7, 8 or 9 from table 1) Glycerine monolaurate  7 6 6 5Glycerine monomyristate 21 18 18 15 Propylene glycol — 10 10 20 Water 5256 46 40

The following experiments were performed to determine the optimumcompositions and method for producing a topical nanoparticulatespironolactone formulation in accordance with the present invention.

Selection of an Optimum Crystalip™ Composition.

Two batches of a Crystalip™ composition were produced as shown in thetable below.

TABLE 3 Reproduction of Crystalip ™ placebo Batch Glycerine GlycerinePropylene glycol number monolaurate monomyristate (PG) Water 4011-001/017% 21% / 72% 4011-001/02 5% 15% 20% 60%

For the first batch, a characteristic exothermic peak was seen duringcooling. At the end of the cooling stage the cream was very viscous butdid not have the shiny appearance typical of a β-crystalline structure.During storage, this shiny appearance started to appear.

For the second batch, an exothermic peak was not observed, however therewas a shiny appearance. The viscosity seemed to be lower than the firstbatch.

A composition with less propylene glycol was then tested (table 4) sincethere were some concerns about irritation resulting from high PGconcentrations. Propylene glycol is useful to retain in the compositionsince it may increase the antimicrobial efficacy of the base and enhancepenetration of active components into the skin therefore. The followingbatches were manufactured:

TABLE 4 Crystalip ™ batches Glycerine Glycerine Propylene Batch numbermonolaurate monomyristate glycol (PG) Water 4011-001/03 5% 15% 10% 70%4011-001/04 6% 18% 10% 66%

For the batch with 20% monoglycerides (MG), viscosity dropped at around40° C., and the mixture became liquid again just after the viscosity hadstarted to increase.

The MG level was therefore increased to 24%. The viscosity also droppedaround 40° C., but viscosity increased again during further cooling. Anexothermic peak was observed at 33° C. and the final cream had a shinyappearance, which means the final β-crystalline structure had beetproduced

Compatibility Tests Between the Surfactant and Crystalip™.

It had previously been determined that Crystalip™ is compatible withMyrj 59 and Span 20, which are both non-ionic surfactants. However,Octowet 70PG is used for the spironolactone nanosuspension, which is ananionic surfactant. Octowet 70PG is also known as Sodium dioctylsulfosuccinate and is a 70% solution (70% DOSS) of water and propyleneglycol

For a cream containing 2% spironolactone, 20% of the spironolactonenanosuspension must be incorporated since the nanosuspension contains10% active spironolactone and 0.5% surfactant. In testing thecompatibility of the Crystalip™ formulation with Octowet solution, 20%Octowet solution was therefore used and replaced part of the waterphase.

The compatibility of Octowet 70PG solution with the chosen Crystalip™formulation (10% PG+24% MG) was tested. Three batches of Crystalip™(table 5) were manufactured in which 20% of a 0.5% Octowet 7OPG solutionwere introduced at different temperatures: 70° C., 50° C. and roomtemperature.

TABLE 5 Crystalip ™ batches including Octowet solution GlycerineGlycerine Octowet Batch mono mono Propylene solution number lauratemyristate glycol (0.5%) Water T ° C.* 4011- 6% 18% 10% 20% 46% 70 001/054011- 6% 18% 10% 20% 46% 50 001/06 4011- 6% 18% 10% 20% 46% room 001/07temp *T ° C.: temperature at which the surfactant solution has beenintroduced.

All three batches produced the exothermic peak and resulted in a shinyappearance. The viscosity of the batches number 05 and 06 wasacceptable, but the viscosity of batch number 07 after introduction ofthe Octowet solution was not sufficient.

It was therefore concluded that the chosen Crystalip™ formula wascompatible with 20% of a 0.5% Octowet solution. The Octowet solution wasbest incorporated at a temperature above the crystallisation point,rather than at room temperature.

Introduction of Spironolactone Nanosuspension in Crystalip™

The composition of the nanosuspension was 10% spironolactone and 0.5%Octowet 70PG.

Particle size of the fresh nanosuspension using laser diffraction(COULTER):D50=0.686

-   -   D90=1.033    -   D99=1.033

Particle size using photon correlation spectroscopy (Zetasizer 3000HS):Z average=476 nm

From the compatibility tests above it was known that incorporatingmaterials at room temperature was not a good option. It was thereforenecessary to investigate heating the nanosuspension. The particle sizebefore and after heating the suspension was measured.

The experiment was carried out as follows:

Two samples of nanosuspension were heated to 50° C. and 70° C.,respectively. The temperature was held for 10 min and then the samplewas cooled back to room temperature. The particle size was measured byphoto correlation spectroscopy and laser diffraction at these timepoints:

-   -   after ultrasonication, before heating (reference)    -   after cooling down (t0)    -   after 24 h (t24 h)

TABLE 6 Determination of size particles by laser diffraction (Coulter)Sample identity Particle size 50° C. - 70° C. - distribution Reference50° C. - t0 70° C. - t0 t24 h t24 h D50 0.579 nm 0.638 nm 0.700 nm 0.634nm 0.694 nm D90 0.740 nm 0.878 nm 1.053 nm 0.861 nm 1.572 nm D99 0.850nm 1.922 nm 1.421 nm 1.902 nm 38.65 nm

The results of Table 6 are also shown in FIGS. 8 and 9.

TABLE 7 Determination of particle size by photon correlationspectroscopy (Zetasizer 3000HS) Sample 50° C. - 70° C. - identityReference 50° C. - t0 70° C. - t0 t24 h t24 h Particles 456.3 469.7538.3 Not done* Not done* size (nm) *the size was found too large bylaser diffraction measurement with PCS was not made

It was shown that the results from heating the nanosuspension show aquite dramatic particle size increase for 70° C., particularly afterwaiting for another 24 h, which suggests some drug had gone intosolution and then re-crystallised. The particle size also increased at50° C., however there did not seem to be so much recrystallisationhappening over the following 24 hours.

It was decided that a very short exposure to 50° C. would be acceptable.It was therefore concluded that the final batch should be prepared asfollows:

Crystalip™ with a reduced water phase would be prepared. Thenanosuspension would be sonicated, but not heated. Once the temperatureof the Crystalip™ reached 50° C., the cold nanosuspension would beadded, leading to a quick temperature decrease and minimisation of heatexposure of the spironolactone suspension. This process would also notinterfere with Crystalip™ crystallisation, which occurs at lowertemperatures.

A batch was prepared according to the above recommendation where coldnanosuspension was added to Crystalip™ at 50° C. (table 8).

TABLE 8 Crystalip ™ batches including spironolactone nanosuspensionGlycerine Batch mono Glycerine Propylene Nano number laurate monomyristate glycol suspension Water 4011- 6% 18% 10% 20% 46% 001/08ac

The batch was successful. The final cream had a good viscosity and ashiny, homogenous appearance.

Viscosity Measurement

Sample ID Composition Bioglan 20% MG-20% PG 4011- 24% MG-10% PG-20%Octowet solution (introduce at 001/05pc 70° C.) 4011- 24% MG-10% PG-20%Octowet solution (introduce at 001/06pc 50° C.) 4011- 24% MG-10% PG-20%Octowet solution (introduce at room 001/07pc temperature) 4011- 24%MG-10% PG-20% nanosuspension 001/08ac 4011- 28% MG 001/13pc 4011- 20%MG-20% PG 001/17pc

The results of the viscosity results of the compositions shown in theabove table are shown in FIGS. 10 and 11.

Tests with the Second Batch of Nanosuspension: 3011-05an1

As the nanosuspension was not fresh for the previous tests it wasdecided to make a new nanosuspension of spironolactone and toincorporate it in Crystalip™ just a few days after the manufacture. Thecomposition of the nanosuspension used was 10% spironolactone and 0.5%Octowet 70PG. It was also investigated whether it was possible tointroduce 30% of nanosuspension instead of 10% in the Crystalip™.

The particle size of the nanosuspension, just after the making, usinglaser diffraction was as follows: (COULTER): D50=0.443

-   -   D90=0.657    -   D99=0.738

Particle size using photon correlation spectroscopy (Zetasizer 3000HS):Z average≈419.3 nm

Two new batches of Crystalip™, 4014-000/01ac and 014-000/02ac were madewith respectively 20% and 30% of nanosuspension in it. The process wasidentical to 4011-001/08ac (incorporation of the nanosuspension at 50°C. during the cooling stage). The nanosuspension was manufactured theday before the making of Crystalip™. For those two batches the pH wasadjusted to the same as the market cream (Spiroderm 5% with a pH=4.16).The two batches are summarised in the following table.

Glycerine Glycerine Batch mono mono Propylene 3011- Citric Sodium numberlaurate myristate glycol 05an1 Water acid Hydroxide 4014- 6% 18% 10% 20%45.5% 0.5% Up to 000/01ac pH = 4.16 4014- 6% 18% 10% 30% 35.5% 0.5% Upto 000/02ac pH = 4.16

The two creams were shiny but the second one 4014-000/02ac seemed tohave a higher viscosity. As the nanosuspension is introduced cold, theviscosity increases more quickly for the batch with 30% ofnanosuspension. Moreover the batch with 30% of nanosuspension seemed tobe less homogeneous because of the increase of the viscosity.

The pH and the density of those batches were measured the next day aftermanufacturing, as shown in the table below.

Batch number pH Density (g/cm3) 4014-000/01ac 4.29 0.989 4014-000/02ac4.22 0.984

Example 2 Size of Spironolactone Particles Before and After Storage

FIGS. 1, 2, and 3 show microscope pictures of Spironolactone accordingto the invention immediately after preparation, after 7 months storageand their comparison to a commercial Spironolactone. The figures containa scale which relates to a distance between each vertical bar of 0.01 mmor 10 micrometers.

The particles shown in FIGS. 1 and 2 are almost too small to see in thelight microscope. There is no particle growth over 7 months storage. Incontrast, the commercial spironolactone “Spiroderm” (FIG. 3) hasSpironolactone crystals present of up to 20 micrometers in size.

Example 3 Flux Studies

The flux through artificial membranes of spironolactone (2%) from ananosuspension formulation incorporated into “Crystalip™” matrix wasmeasured in a Franz cell set-up and compared with 2% w/w spironolactonein Aqueous cream B.P. as a comparator.

Material Supplier Crystalip ™ spironolactone SkyePharma, Switzerlandnanosuspension 2% Lot no. 4014-000/06atc Spironolactone Lot no. 510/0Aqueous cream B.P. Hillcross, UK Lot no. 28076 Ethanol VWR InternationalLtd., UK AnalaR grade Sodium dihydrogen phosphate dihydrate MerckeurolabLot no. L298 Deionised water Elga Ltd., UK Acetonitrile HPLC gradeRathburns Chemicals Ltd., UK Regenerated cellulose MembraneNBS-Biological, UKMethods for Flux Studies

The Crystalip™ formulation was supplied by SkyePharma AG. A commercialSpironolactone comparator was not available any more at the time of theexperiments. Therefore a comparator in a standard cream matrix wasprepared as follows. Briefly, 100 mg of Spironolactone powder wasaccurately weighed and mixed with 4.90 g Aqueous cream B.P. in order toobtain a 2% w/w non-nanoparticulate spironolactone in Aqueous creamformulation.

In-vitro Diffusion Studies

Ethanol: Phosphate ‘buffer’ (20:80 v/v, pH 4.5) was used as the receiverfluid in order to maintain stability of spironolactone and sinkconditions. The artificial membrane used was regenerated cellulosemembrane.

Franz Cell Diffusion Studies

Individually calibrated Franz diffusion cells with an averagediffusional surface area of 0.56±0.03 cm2 and an average receiver volumeof 1.83±0.02 ml were used to conduct the diffusion experiments. TheSpectra/Por® cellulose membranes were cut to appropriate size andimmersed in deionised water for 30 min to remove the preservative (0.1%sodium azide), wiped with tissue to remove surface liquid and mountedonto the Franz cells. The receiver fluid was incorporated into the Franzcell, stirred constantly with a magnetic stirrer and maintained at 32°C. The membranes were allowed to equilibrate with the receiver phase for30 min before applying the formulations. Each formulation (200 μl) wasapplied onto the membrane surface using a positive displacementFinnpipette®. Five sampling times were investigated (1, 2, 4, 6 and 8 h)whereby 200 μl of the receiver fluid was carefully withdrawn from thearm of the Franz cell; each sample removed was replaced by an equalvolume of fresh pre warmed (32° C.) receiver fluid. Throughout theexperiment, any losses in receiver fluid due to evaporation from theFranz cell were replaced to maintain a constant volume. Samples wereanalysed via HPLC using chromatographic conditions as follows:

Column: Hypersil 3 μm Phenyl BDS column (s/no. 182862) Column length:150 × 4.60 mm Column temperature: 30° C. Mobile phase: 50 mM Phosphatebuffer:acetonitrile (70:30 v/v) Flow rate: 1.0 ml/min UV wavelength: 238nm Injection volume: 10 μl Run time: 15 minPreparation of Spironolactone Standard Curves

Spironolactone standards were prepared in receiver fluid and calibrationcurves were constructed in the range 0.93-59.2 μg/ml. Calibration curveswith r2>0.999 were considered acceptable and a typical curve isillustrated in FIG. 4.

Data Analysis

The amount of spironolactone in the receiver fluid was corrected forsample removal. The cumulative amount of spironolactone permeated perunit membrane surface was plotted against the square root of time andthe slope of the linear portion of the graph was estimated as the steadystate flux. A Student's t-test was employed to statistically determineany significant difference in release of spironolactone from the 2%spironolactone nanosuspensions and 2% spironolactone Aqueous cream.

Results

FIG. 5 shows a graphical representation of the mean cumulative amount ofspironolactone permeated per unit area (μg/cm2) from the twospironolactone formulations. These profiles show steady state flux forboth formulations. The release rate of spironolactone from thespironolactone nanosuspension (2% w/w) was shown to be significantlyfaster than the aqueous cream formulation (p=0.08).

Example 4 Zone of Inhibition Assay

The antimicrobial action of two spironolactone formulations, namelyspironolactone nanosuspension 2% w/w and spironolactone coarse 2% wlw,and their respective placebos were compared. A 2% w/w spironolactone inAqueous cream B.P. formulation (in-house) was used as a comparator. Thefollowing materials were used.

Material Supplier Staphylococcus epidermidis Oxoid Ltd, UK. (ATCC 12228)Pseudomonas acne Central Public Health Laboratory, UK (NTC 737) S&SAntibiotic-Assay Aldrich Chemical company, USA discs(filter paper), diam¼ inch Aqueous cream B.P. Hillcross, UK Lot no. 28076 SpironolactoneSkyePharma, Switzerland Lot no. 510/0Methods

The Crystalip™ spironolactone and placebo formulations were prepared asshown in the table below. Since a commercial Spironolactone comparatorwas not available any more, a comparator in a standard cream matrix wasprepared as follows. Briefly, 100 mg of Spironolactone powder wasaccurately weighed and mixed with 4.90 g Aqueous cream B.P. to obtain a2% wlw non-nanoparticulate spironolactone in Aqueous cream.

The batch size is 500 g, manufactured with a lab reactor IKA-LR1000.2.

4014-000/06atc Crystalip ™ Composition nanosuspension 2% MG(monoglycerides) 24 PG (propylene glycol) 10 Nanosuspension - 10% spiro20 Spironolactone coarse / Octowet solution 0.5% w/w / Citric acid 0.1Sodium Hydroxide up to pH 4.16 Water PPI 45.9 Physical properties pH4.31 Density (g/ccm) 0.900 Viscosity (cP) 20 rpm 42′327 50 rpm 23′780

20±1 mg of each of the samples listed below were carefully transferredonto the surface of ¼ inch antibiotic assay discs.

-   1 SkyePharma AG, Crystalip™, Spironolactone nanosuspension 2% w/w    4014-000/06atc-   2 2% w/w Spironolactone in Aqueous cream BP

The antibiotic discs coated with each of the formulations were placedonto the surface of the organism seeded agar plates using a pair ofsterile forceps.

The S. epidermidis plates were incubated for 24 h at 37° C.

The P. acne plates were incubated in the anaerobic jars and incubatedfor 72 h.

Zones of inhibition were measured using a pair of calipers.

Results

FIGS. 6 and 7 show the mean zone of inhibition for both formulationsusing seeded S. epidermidis and P. acnes plates, respectively. TheCrystalip™ formulation with Spironolactone nanosuspension exhibited aconsiderable effect against those acne-related bacteria Aqueous creamB.P. with 2% w/w spironolactone (comparator) showed no zones ofinhibition.

The base matrix therefore adds an antibacterial effect (on S.epidermidis and P. acnes) to the formulation, which is not due to theSpironolactone. The formulation does not contain any furtherantibiotics, or preservatives. The comparator product, which shows zeroantibiotic effect on these acne-related microorganisms is preserved withphenoxyethanol. The applicants product may therefore improve acnethrough both the hormone activity of the drug and the antibacterialefficacy of the matrix in which it is contained.

1. A stable topical nanoparticulate spironolactone formulationcomprising nanoparticles of spironolactone incorporated into acrystalline network of polar lipids, wherein the nanoparticles ofspironolactone have a mean diameter measured by photon correlationspectroscopy in the range of from about 300 nm to about 900 nm.
 2. Theformulation according to claim 1, comprising nanoparticles having a meandiameter, measured by photon correlation spectroscopy, in the range offrom about 400 nm to about 600 nm.
 3. The formulation according to claim1, wherein the lipid has a crystallization temperature of between 20° C.and 100° C.
 4. The formulation according to claim 1, wherein thecrystalline network of polar lipids is formed from 13 crystals of amonoglyceride of a fatty acid having 12-18 carbon atoms, or ascorbic,phosphate or lactic esters of fatty acids or of monoglycerol ethers, ormixtures thereof.
 5. The formulation according to claim 4, wherein themonoglyceride is 1-monolaurin, 1-monomyristin, 1-monopalmitin, or1-monostearin, or a mixture of two or more thereof.
 6. The formulationaccording to claim 1, wherein crystalline network structures of polarlipids are formed within a polar liquid.
 7. The formulation according toclaim 6, wherein the polar liquid is selected from water, glycerol,ethylene glycol, propylene glycol, or mixtures thereof.
 8. A method oftreating one or more of acne, hirsutism, androgenic alopecia, orrosacea, comprising topically applying to a subject in need thereof thenanoparticulate spironolactone formulation according to claim
 1. 9. Theformulation according to claim 1, wherein active drug is incorporated inthe form of a nanosuspension.
 10. The formulation according to claim 9,wherein the nanosuspension is an aqueous nanosuspension.
 11. Theformulation according to claim 10, wherein the nanosuspension comprisesa stabilizer.
 12. The formulation according to claim 11, wherein thestabilizer is sodium docusate.
 13. A method of treating a condition thatresponds to anti-androgens comprising: administering a stablenanoparticulate spironolactone formulation according to claim 1 to apatient in need of such treatment, wherein said condition is acne,hirsutism, androgenic alopecia, or rosacea.
 14. A process for thepreparation of a stable topical nanoparticulate spironolactoneformulation comprising: dispersing nanoparticulate spironolactone into amixture of polar lipids and a polar liquid at a temperature below thetransition temperature of the lipid but above the temperature at whichthe lipid crystalline structure is fully formed.
 15. A method oftreating a condition that responds to anti-androgens, comprisingadministering a stable topical nanoparticulate spironolactoneformulation comprising nanoparticles of spironolactone incorporated intoa crystalline network of polar lipids in an amount effective to treatthe condition, wherein said condition is acne, hirsutism, androgenicalopecia, or rosacea, and wherein the nanoparticles of spironolactonehave a mean diameter measured by photon correlation spectroscopy in therange of from about 300 nm to about 900 nm.
 16. The method according toclaim 8, wherein spironolactone active drug is incorporated into theformulation in the form of a nanosuspension.
 17. The method according toclaim 13, wherein spironolactone active drug is incorporated into theformulation in the form of a nanosuspension.
 18. The formulationaccording to claim 1, wherein said nanoparticles do not grow followingseven months in storage at room temperature.
 19. The method according toclaim 14, wherein said nanoparticles do not grow following seven monthsin storage at room temperature.
 20. The method according to claim 15,wherein said nanoparticles do not grow following seven months in storageat room temperature.